Physiological Media Research Articles

CD8+ Tcell metabolic rewiring defined by scRNA-seq identifies a critical role of ASNS expression dynamics in Tcell differentiation.

T cells dynamically rewire their metabolism during an immune response. We applied single-cell RNA sequencing to CD8+ Tcells activated and differentiated invitro in physiological medium to resolve these metabolic dynamics. We identify a differential time-dependent reliance of activating Tcells on the synthesis versus uptake of various non-essential amino acids, which we corroborate with functional assays. We also identify metabolic genes that potentially dictate the outcome of Tcell differentiation, by ranking them based on their expression dynamics. Among them, we find asparagine synthetase (Asns), whose expression peaks for effector Tcells and decays toward memory formation. Disrupting these expression dynamics by ASNS overexpression promotes an effector phenotype, enhancing the anti-tumor response of adoptively transferred CD8+ Tcells in a mouse melanoma model. We thus provide a resource of dynamic expression changes during CD8+ Tcell activation and differentiation, and identify ASNS expression dynamics as a modulator of CD8+ Tcell differentiation.

Fluorescent carbon quantum dots-based prodrug nanosponges with outstanding tumor-specific drug delivery and imaging

• Fluorescent carbon quantum dots-based nanotheranostics were designed. • Longer linkers between the building blocks of CQDs could boost the drug content. • Hydrophobic linkers could minimize the premature DOX leakage. Carbon quantum dots (CQD)-based nanosponges have been widely designed for tumor theranostic application recently. However, the drug content is usually lower with the shorter dynamic covalent linkers while the longer hydrophilic dynamic covalent linkers led to a significant premature drug leakage. Here, a hydrophobic linear reducible-responsive linker with two terminal aldehyde group was designed to crosslink the hydrazine-functionalized PEGylated CQDs (Hy-CQD-PEG), resulting in Hy-CQDss-PEG nanosponges. After doxorubicin (DOX) conjugation, the DOX-Hy-CQDss-PEG prodrug nanosponges were obtained with DOX content of 18.76 % and hydrodynamic diameter of 140 nm, respectively. The DOX-Hy-CQDss-PEG nanosponges were stable in the normal physiological medium with very low drug leakage and fluorescence, while they could disintegrate in the tumor intracellular microenvironment, releasing DOX and the hydrazine-functionalized CQDs for theranostic application.

Water soluble octa-imidazolium zinc phthalocyanine for nucleus/nucleolus cell fluorescence microscopy and photodynamic therapy.

A poly-cationic theranostic macrocycle was developed to perform confocal microscopy imaging and photodynamic therapy studies on a model of melanoma cancer, one of the most aggressive cancer. Hence, an octa-imidazolium zinc phthalocyanine was conveniently synthesized in large amount in three steps in a 44% overall yield: upon double nucleophilic aromatic substitution, cyclo-tetramerization and quaternization reactions. Such an octa-cationic molecule was readily soluble in physiological media, reaching concentrations beyond 1mM. It showed fluorescence properties in aqueous medium (ΦF = 0.31) with no noticeable aggregation, spectroscopy studies showed. In vitro confocal fluorescence microscopy studies carried out on murine melanoma model (B16F10 cells) showed that the fluorophore was mainly located in the cell nucleolus, an organelle of interest for the treatment of cancer. The anticancer photodynamic potential of the octa-cationic photosensitizer could be measured (IC50 = 5.4µM) using the MTS viability assay. Both fluorescence microscopy studies and photodynamic studies demonstrate the octa-cationic molecule is theranostic and could be further developed for future photodynamic diagnosis (PDD) and photodynamic inactivation of micro-organisms (PDI).

The impact of cell culture media on the interaction of biopolymer-functionalized gold nanoparticles with cells: mechanical and toxicological properties

Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.

Physiological Media in Studies of Cell Metabolism.

Changes in cell metabolism accompany the development of a wide spectrum of pathologies including cancer, autoimmune, and inflammatory diseases. Therefore, usage of inhibitors of metabolic enzymes are considered a promising strategy for the development of therapeutic agents. However, the investigation of cellular metabolism is hampered by the significant impact of culture media, which interfere with many cellular processes, thus making cellular models irrelevant. There are numerous reports that show that the results from in vitro systems are not reproduced in in vivo models and patients. Over the last decade a novel approach has emerged, which consists of adaptation of the culture medium composition to that closer to the composition of blood plasma. In 2017‒2019, two plasma-like media were proposed, Plasmax and HPLM. In the review, we have summarized the drawbacks of common media and have analyzed changes in the metabolism of cells cultivated in common and plasma-like media in normal and pathological conditions.

In Vitro Degradation and In Vivo Biocompatibility of Strontium-Doped Magnesium Phosphate-Reinforced Magnesium Composites.

Magnesium is projected for use as a degradable orthopedic biomaterial. However, its fast degradation in physiological media is considered as a significant challenge for its successful clinical applications. Bioactive reinforcements containing Mg-based composites constitute one of the promising approaches for developing degradable metallic implants because of their adjustable mechanical behaviors, corrosion resistance, and biological response. Strontium is a trace element known for its role in enhancing osteoblast activity. In this study, bioactive SrO-doped magnesium phosphate (MgP)-reinforced Mg composites containing 1, 3, and 5 wt % MgP were developed through the casting route. The influence of the SrO-doped MgP reinforcement on degradation behaviors of the composites along with its cell-material interactions and in vivo biocompatibility was investigated. The wt % and distribution of MgP particles significantly improved the mechanical properties of the composite. HBSS immersion study indicated the least corrosion rate (0.56 ± 0.038 mmpy) for the Mg-3MgP composite. The higher corrosion resistance of Mg-3MgP leads to a controlled release of Sr-containing bioactive reinforcement, which eventually enhanced the cytotoxicity as measured using MG-63 cell-material interactions. The in vivo biocompatibility of the composite was evaluated using the rabbit femur defect model. Micro-computed tomography (μ-CT) and histological analysis supported the fact that Mg-3MgP maintained its structural integrity and enhanced osteogenesis (50.36 ± 2.03%) after 2 months of implantation. The results indicated that the Mg-MgP composite could be used as a degradable internal fracture fixation device material.

Anti-inflammatory effect evaluation of naringenin and its incorporation into a chitosan-based film for transdermal delivery

Naringenin is a bioflavonoid mainly found in citrus fruits. It presents many pharmacological benefits, including a remarkable anti-inflammatory activity, but its oral bioavailability is poor. To overcome this drawback, this work proposes a transdermal administration of such bioflavonoid, considering its use in the chronic treatment of inflammatory conditions. For this, it aims to develop a chitosan-based film that guarantees a consistent transdermal delivery of the drug. First, naringenin's in vitro anti-inflammatory effect on T-cell proliferation was evaluated, followed by research on the modulation of gene expression for inflammatory factors in peripheral blood mononuclear cells. Chitosan films were then prepared and characterized. Afterward, naringenin release profile from a selected film was determined as well as the drug permeation across porcine skin provided by the film. Naringenin induced the expression of the anti-inflammatory factors IL-10 and TGF-β1 while inhibiting the expression of the pro-inflammatory cytokine IL-1β and limiting T-cell proliferation. The chitosan film was successfully developed, and the drug was progressively released to the physiological media following both first order and Korsmeyer-Peppas kinetics. When topically applied, the chitosan film guaranteed a constant and continuous diffusion of naringenin across the skin over 72 h. Indeed, the permeation flux of naringenin was 0.30 ± 0.01 µg/cm2/h, which means a concentration in the receptor solution 14-fold (p < 0.05) higher than that provided by the drug solution. Thus, the chitosan film represents a promising transdermal alternative for the long-term treatment of inflammatory conditions using naringenin.

A simple, robust and scalable route to prepare sub-50 nm soft PDMS nanoparticles for intracellular delivery of anticancer drugs

Soft nanoparticles (NPs) have recently emerged as a promising material for intracellular drug delivery. In this regard, NPs derived from polydimethylsiloxane (PDMS), an FDA approved polymer can be a suitable alternative to conventional soft NPs due to their intrinsic organelle targeting ability. However, the available synthesis methods of PDMS NPs are complicated or require inorganic fillers, forming composite NPs and compromising their native softness. Herein, for the first time, we present a simple, robust and scalable strategy for preparation of virgin sub-50 nm PDMS NPs at room temperature. The NPs are soft in nature, hydrophobic and about 30 nm in size. They are stable in physiological medium for two months and biocompatible. The NPs have been successful in delivering anticancer drug doxorubicin to mitochondria and nucleus of cervical and breast cancer cells with more than four-fold decrease in IC50 value of doxorubicin as compared to its free form. Furthermore, evaluation of cytotoxicity in reactive oxygen species detection, DNA fragmentation, apoptosis-associated gene expression and tumor spheroid growth inhibition demonstrate the PDMS NPs to be an excellent candidate for delivery of anticancer drugs in mitochondria and nucleus of cancer cells.

Rapid microwave synthesis of magnetic nanoparticles in physiological serum

Abstract Superparamagnetic Iron Oxide Nanoparticles (SPIONs) are more and more used in biomedical applications such as therapy (treatment for certain cancers, hyperthermia), diagnostic (contrast agent for Magnetic Resonance Imaging) or both. For these applications, SPIONs must be stable in an aqueous solution, monodisperse, with a narrow size distribution and without aggregation. To obtain these nanoparticles, a microwave process is carried out in this study as an easy, fast and reproducible synthesis method. Currently, in the literature, most synthesis of SPIONs are in ultra-pure water or another solvent. To consider the use of SPIONs in biomedical applications, it is essential to ensure the preservation of the physico-chemical parameters of the nanoparticles in the physiological medium to validate a synthesis process. With this objective, this study reports a comparison between the SPIONs synthesis in ultra-pure water and the SPIONs direct synthesis in a physiological serum (containing NaCl). To complete this comparison, the dispersion of SPIONs in physiological serum after an elaboration in ultra-pure water is reported. Characterizations of these different SPIONs samples are carried out to determine the physico-chemical parameters and magnetic properties. SPIONs are characterized by Transmission Electronic Microscopy, Dynamic Light Scattering, X-Ray Diffraction, Raman spectroscopy and magnetic measurements. Finally, to check if SPIONs can be used as contrast agent for MRI, a relaxometry measurement is performed.

Mechanical, tribological and electrochemical behavior of Zr-based ceramic thin films for dental implants

To determine the possibility of using new thin films architectures as biocompatible materials, an experimental and computational study was performed to evaluate the mechanical, tribological, and corrosion properties in simulated physiological media (saliva and blood plasma) of Zr, ZrN, and ZrN/Zr coatings, deposited by PVD magnetron sputtering. The crystalline structure and chemical composition were well correlated with high resistance to plastic deformation, wear, and corrosion, making these materials excellent candidates for functionalizing and protecting dental prostheses. The predominant wear mechanism under consideration was abrasion, which was reduced when using ceramic ZrN coating as a base for the superficial Zr thin film. When exposed to simulated body fluids, these materials exhibited high corrosion resistance, which was demonstrated by potentiodynamic measurements. These results are consistent with those predicted by Density Functional Theory computational models, which showed that electron transfer associated with the wear mechanism is kinetically impeded, as a consequence of the large energy barriers for this process associated with the adsorption of the molecular species on the ZrN surface. Additionally, calculated adsorption energies indicated that urea (from the simulated saliva solution) interacts strongly with the surface. This interaction was associated to the formation of passivating protective layers, which is a key mechanism to protect against corrosion, acting in synergy with the kinetic barriers.

Physiological media advance cell culture experiments

The metabolism plays a fundamental role in cellular signaling pathways, but commonly used cell culture media do not reflect physiological metabolite concentrations. The metabolic control hub mammalian target of rapamycin complex 1 (mTORC1) kinase is an illuminating example that it is about time to advance our cell culture to become more physiological and relevant.

Advanced transcriptomic analysis reveals the role of efflux pumps and media composition in antibiotic responses of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen and major cause of hospital-acquired infections. The virulence of P. aeruginosa is largely determined by its transcriptional regulatory network (TRN). We used 411 transcription profiles of P. aeruginosa from diverse growth conditions to construct a quantitative TRN by identifying independently modulated sets of genes (called iModulons) and their condition-specific activity levels. The current study focused on the use of iModulons to analyze the biofilm production and antibiotic resistance of P. aeruginosa. Our analysis revealed: (i) 116 iModulons, 81 of which show strong association with known regulators; (ii) novel roles of regulators in modulating antibiotics efflux pumps; (iii) substrate-efflux pump associations; (iv) differential iModulon activity in response to beta-lactam antibiotics in bacteriological and physiological media; (v) differential activation of ‘Cell Division’ iModulon resulting from exposure to different beta-lactam antibiotics and (vi) a role of the PprB iModulon in the stress-induced transition from planktonic to biofilm lifestyle. In light of these results, the construction of an iModulon-based TRN provides a transcriptional regulatory basis for key aspects of P. aeruginosa infection, such as antibiotic stress responses and biofilm formation. Taken together, our results offer a novel mechanistic understanding of P. aeruginosa virulence.

Potential of Biodegradable Magnesium Alloys for Medical Applications

Biodegradability of magnesium alloys in physiological media is important for material use in implant manufacture industry. Two industrial Mg alloys ZQ71 and ZQ63 were investigated. Optical microscopy was used to put in evidence microstructure. The conclusions are correlated with obtained results after scanning electron microscopy investigations coupled with energy dispersive X-ray spectroscopy. The evaluation of the hydrogen released rate was analyzed in laboratory made simulated body fluid (SBF) and Hanks’ solution at 37°C for 10 days. Different degradation rates are obtained, and it can be concluded that they depend on chemical composition of the alloys and on immersion time of the samples in different physiological solutions.

Red-emitting polyaniline-based nanoparticle probe for pH-sensitive fluorescence imaging.

Fluorescent probes based on semiconducting polymer nanoparticles (NPs) such as polyaniline (PANI) usually require external fluorophore doping to provide fluorescence function. Direct use of PANI-based NPs for bioimaging applications has been limited by PANI's weak blue fluorescence and aggregation-induced quenching in physiological medium. In this report, we developed a facile solid-state synthesis method to produce fluorescent polyaniline nanoparticles (FPNs) that are not only water-soluble but also exhibit high intensity and pH-sensitive red fluorescence. The FPNs showed high photoluminescence quantum yield (PLQY) of 19.3% at physiological pH, which makes FPNs ideal for application as fluorescent nanoprobes in bioimaging. Moreover, we performed an in-depth study of photoluminescence dependence on pH and the phenomena of exciton-polaron quenching at low pH was highlighted. We also found that the ratio of emission intensity at 600nm and 650nm increased from 0.04 to 1.65 as pH was raised from 2.6 to 11.8, which could find its application in ratiometric pH sensing. FPNs exhibited excellent biocompatibility with >85% cell viability for fibroblasts NIH/3T3 and prostate cancer 22RV1 cells even at concentrations as high as 1000μg/mL. In addition, fluorescence microscopy demonstrated concentration-dependent red fluorescence in the cytoplasm owing to the cellular uptake of FPNs in prostate cancer cells.

Nanosized magnetite modified with poly(ethylene glycol) for efficient sorption of L-lysine-α-oxidase from the culture fluid

Fe3O4@PEG have been proposed for sorption of L-lysine-α-oxidase (LO) from the culture fluid of Trichoderma harzianum Rifai F-180 for the first time. To synthesize the Fe3O4@PEG nanoparticles, an original method based on aqueous biphasic systems has been developed. The PEG-modified magnetite provide a high sorption ability towards LO in contrast to the non-modified Fe3O4 synthesized by the traditional precipitation method. The morphology and structure of the prepared nanoparticles were characterized by TEM, FTIR and XRD. The data on magnetic properties and stability in physiological media are presented. The synthesized nanoparticles ensure quantitative sorption and desorption of LO during at least 3 cycles.

Antioxidant activity and redox properties of cis-2,4,5-tris(hydroxyaryl)imidazolines

New effective antioxidants of cis-2,4,5-tris(hydroxyaryl)imidazoline type were assembled from aromatic aldehydes and ammonia. Their antioxidant activity was measured against hydroxyl radical and superoxide radical anion in physiological media, and oxidation potentials were measured by cyclic voltammetry. The lead compound bearing 3,4,5-hydroxyphenyl moieties showed activity 3.5 times higher compared to Trolox.

Surface stabilization determines macrophage uptake, cytotoxicity, and bioactivity of curcumin nanocrystals

Pharmaceutical nanocrystals represent a promising new formulation that combines the benefits of bulk crystalline materials and colloidal nanoparticles. To be applied in vivo, nanocrystals must meet several criteria, namely colloidal stability in physiological media, non-toxicity to healthy cells, avoidance of macrophage clearance, and bioactivity in the target tissue. In the present work, curcumin, a naturally occurring poorly water-soluble molecule with a broad spectrum of bioactivity has been considered a candidate substance for preparing pharmaceutical nanocrystals. Curcumin nanocrystals in the size range of 40–90 nm were prepared by wet milling using the following combination of steric and ionic stabilizers: Tween 80, sodium dodecyl sulfate, Poloxamer 188, hydroxypropyl methylcellulose, phospholipids (with and without polyethylene glycol), and their combination. Nanocrystals stabilized by a combination of phospholipids enriched with polyethylene glycol proved to be the most successful in all evaluated criteria; they were colloidally stable in all media, exhibited low macrophage clearance, and proved non-toxic to healthy cells. This curcumin nanoformulation also exhibited outstanding anticancer potential comparable to commercially used cytostatics (IC50 = 73 µM; 24 h, HT-29 colorectal carcinoma cell line) which represents an improvement of several orders of magnitude when compared to previously studied curcumin formulations. This work shows that the preparation of phospholipid-stabilized nanocrystals allows for the conversion of poorly soluble compounds into a highly effective “solution-like” drug delivery system at pharmaceutically relevant drug concentrations.

Carboxylate functionalized NaDy(MoO4)2 nanoparticles with tunable size and shape as high magnetic field MRI contrast agents

Uniform sodium-dysprosium double molybdate (NaDy(MoO4)2) nanoparticles having different morphologies (spheres and ellipsoids) and tunable size have been synthesized for the first time in literature. The procedure is based on a homogeneous precipitation process at moderated temperatures (≤220 °C) from solutions containing appropriated precursors dissolved in ethylene glycol-water mixtures, in the absence (spheres) or the presence (ellipsoids) of tartrate anions. The effects of the morphological characteristics (size and shape) of the nanoparticles on the magnetic relaxivity at high field (9.4 T) have been evaluated finding that the latter magnitude was higher for the spheres than for the ellipsoids, indicating their better suitability as contrast agents for high-field magnetic resonance imaging. Such nanoparticles have been successfully coated with polymers bearing carboxylate functional groups through a layer-by-layer process, which improves the colloidal stability of the nanoparticles in physiological media. It has been also found that the coating layer had no significant effects on the nanoparticles relaxivity and that such coated nanoparticles exhibited a high biocompatibility and a high chemical stability. In summary, we have developed NaDy(MoO4)2 based bioprobes which meet the required criteria for their use as contrast agents for high-field magnetic resonance imaging.

A Gallic Acid-Doped Polypyrrole Coating with Anticorrosion and Antibacterial Properties on Magnesium Alloy.

Magnesium (Mg) and its alloys exhibit great potential as biomedical implants due to their excellent biological performance and mechanical properties. However, their clinical applications are limited by their rapid corrosion rate in physiological media and the risk of implant-associated infections. Herein, a multifunctional polypyrrole/gallic acid (PPy/GA) coating was deposited on an AZ31 Mg alloy substrate by electrochemical polymerization to enhance simultaneously the corrosion resistance and antibacterial properties of the Mg alloy. Electrochemical and in vitro immersion tests demonstrated that the anticorrosion performance of the Mg alloy was significantly improved with the PPy/GA coating. The thiazolyl blue tetrazolium bromide (MTT) assay and live-dead staining of L929 cells indicated the acceptable cytocompatibility of the PPy/GA coating. In vitro antibacterial tests revealed a remarkable enhancement in the antibacterial activity of the PPy/GA-coated Mg alloy compared with the PPy-coated material and the bare Mg alloy.

The evaluation of ADME and pharmacokinetic properties of decoquinate derivatives for the treatment of malaria.

The emergence of Plasmodium falciparum (Pf) parasite strains tolerant of the artemisinin component and resistant to the other drug component in artemisinin combination therapies (ACTs) used for treatment now markedly complicates malaria control. Thus, development of new combination therapies are urgently required. For the non-artemisinin component, the quinolone ester decoquinate (DQ) that possesses potent activities against blood stage Pf and acts on a distinct target, namely the Pf cytochrome bc 1 complex, was first considered. However, DQ has poor drug properties including high lipophilicity and exceedingly poor aqueous solubility (0.06 μg/ml), rendering it difficult to administer. Thus, DQ was chemically modified to provide the secondary amide derivative RMB005 and the quinoline O-carbamate derivatives RMB059 and RMB060. The last possesses sub-nanomolar activities against multidrug resistant blood stages of Pf, and P. berghei sporozoite liver stages. Here we present the results of ADME analyses in vitro and pharmacokinetic analyses using C57BL/6 mice. The amide RMB005 had a maximum mean whole blood concentration of 0.49 ± 0.02 µM following oral administration; however, the area under the curve (AUC), elimination half-life (t1/2) and bioavailability (BA) were not significantly better than those of DQ. Surprisingly, the quinoline O-carbamates which can be recrystallized without decomposition were rapidly converted into DQ in human plasma and blood samples. The maximum concentrations of DQ reached after oral administration of RMB059 and RMB060 were 0.23 ± 0.05 and 0.11 ± 0.01 µM, the DQ elimination half-lives were 4.79 ± 1.66 and 4.66 ± 1.16 h, and the DQ clearance were 19.40 ± 3.14 and 21.50 ± 3.38 respectively. Under these assay conditions, the BA of DQ could not be calculated Overall although RMB059 and -060 are labile in physiological medium with respect to the DQ parent, the potential to apply these as prodrugs is apparent from the current data coupled with their ease of preparation.